Reliable personal glucose monitoring devices are of critical importance to diabetics. Currently, determination of blood glucose is achieved using hand-held devices, which require invasive pin-prick sampling of blood glucose levels. These tests are painful, inconvenient, need to be performed several times per day, do not provide a continuous measurement and risk missing important fluctuations in glucose levels.
Subcutaneous, implantable sensors that remain in permanent contact with interstitial fluid, or contact lens sensors that measure glucose levels in tear fluid, may be used to perform non-invasive blood glucose monitoring. Enzyme-based systems that are currently used for invasive blood glucose monitoring using glucose oxidase, are unlikely to be suitable for this purpose. Their relatively high cost, inadequate stability and lack of sterilisability complicate their use in vivo. Therefore, glucose-responsive devices are required that mimic the selectivity and specificity of enzyme-based systems, whilst offering a more robust and sterilisable detection system.
Boronic acids are weak Lewis acids; the electron-deficient boron atom exists in either an uncharged planar trigonal form or, when interacting with a strong base such as hydroxide, a charged tetrahedral form. Both forms may covalently bind vicinal diols with the reversible formation of a boronic acid diester and the liberation of two equivalents of water. The pKa of the boronic acid diester is significantly reduced compared to the boronic acid and thus, at suitable pH, binding of vicinal diols results in the formation of the charged boronate ester. Due to favourable stereochemistry, the binding constant is significantly greater when the boronic acid is in the tetrahedral form (Bosch et al., 2004). A set of coupled equilibria resulting from these interactions is shown in FIG. 1. The pKa(1) of phenylboronic acid has been reported to be around 8.9; thus, the tetrahedral boronate, which binds cis-diols much more readily than the trigonal form, is present at very low concentrations at physiological pH. Development of boronic acid ligands for physiological glucose detection has focused on reducing the pKa via synthetic modification of the boronic acid receptor (Wiskur et al., 2001). It is known that a charge stabilisation mechanism helps stabilize the formation of the boronate-vicinal diol diester (Badugu et al, 2005).
Optical measurements offer particular advantages for non-invasive glucose monitoring because they circumvent the practical difficulties associated with electrical connections to the sensor device. However, both chromophore and fluorophore-functionalised receptors are susceptible to photochemical instability. In addition, fluorescence measurements in biological media can be complicated by background fluorescence, low fluorophore solubility and oxygen interference.
Polymeric sensors can be fabricated that are optically responsive to receptor-ligand binding via changes to their diffraction properties. Glucose-responsive have been reported, based on phenylboronic acid-functionalised polymers (Alexeev et al., 2003). However, the fabrication of these materials is time-consuming and may not be suitable for mass manufacture.
WO95/26499 discloses a holographic sensor, based on a volume hologram. The sensor comprises a holographic element, the element comprising an analyte-sensitive holographic support medium having an optical transducing structure disposed throughout its volume. Because of this physical arrangement of the transducer, the optical signal generated by the sensor is very sensitive to volume changes or structural rearrangements taking place in the analyte-sensitive matrix as a result of interaction or reaction with the analyte. For example, a sensor comprising a gelatin-based holographic medium may be used to detect trypsin. Trypsin acts on the gelatin medium, irreversibly destroying the integrity of the holographic support medium.
WO03/087899 describes a method for the continuous detection of an analyte in a fluid involving the use of a holographic sensor. The support medium of the sensor comprises a group which is capable of reacting reversibly with the analyte. Thus, when fluid is passed over the holographic element, any analyte present can be detected continuously.
In particular, WO03/087899 describes how a holographic sensor formed by the polymerisation of monomers including vinyiphenylboronic acid may be used to detect glucose. The pendant phenylboronic acid groups can react reversibly with a vicinal diol group of glucose, resulting in swelling of the holographic support medium.
WO04/081624 is based on the discovery of a class of phenylboronic acid derivatives which allow for the detection of glucose and other vicinal diol-containing analytes across a wide range of pH values. These phenylboronic acids can be modified to promote formation of a more reactive tetrahedral conformation at low pH values.
For example, the phenyl group may comprise one or more electron-withdrawing substituents which, by mediating their electronic effects through the phenyl ring, promote formation of RB(OH)3−. As another example, a substituent may be capable of forming an intramolecular bond with the boron atom, forcing the boronate into a substantially tetrahedral conformation. Judicious selection of substituents allows the responsiveness of the sensor to be optimised with respect to a particular set of detection conditions.
WO04/081624 discloses a sensor for the detection of an analyte comprising a vicinal diol moiety, which comprises a holographic element comprising a medium and a hologram disposed throughout the volume of the medium, wherein an optical characteristic of the element changes as a result of a variation of a physical property occurring throughout the volume of the medium, and wherein the medium comprises a polymer comprising a group of formula (i)
wherein
n is 0, 1, 2, 3 or 4;
each X (if present) is independently an atom or group which, via an electronic effect, promotes formation of a tetrahedral geometry about the boron atom; and
Y is a spacer which, when n is 0 or otherwise optionally, is an atom or group which, via an electronic effect, promotes formation of a tetrahedral geometry about the boron atom.
Such a sensor can be used in a method for the detection of an analyte comprising a vicinal diol moiety in a fluid, which comprises contacting the fluid with the holographic element and detecting any change of the optical characteristic of the element. The analyte may comprise a plurality of vicinal diol moieties; examples of such analytes include glucose and tartaric acid.
WO04/081624 also discloses that in addition to a compound of formula (i), the monomers may include (meth)acrylamide and/or (meth)acrylate-derived co-monomers. In particular, the monomer HEMA (hydroxyethyl methacrylate) is readily polymerisable and cross-linkable. PolyHEMA is a versatile support material since it is swellable, hydrophilic and widely biocompatible. The monomers may also include co-monomers having groups which are capable of intermolecular electron-donation, for example secondary or tertiary amines.
Glucose-responsive holographic sensors, functionalised with 4-vinylphenylboronic acid (4-VBPA), based on acrylamide co-polymer hydrogel films are described elsewhere in more detail (Kabilan et al., 2004a). Upon glucose binding, the conversion of the electron-deficient sp2-hydridised boron to the charged boronate causes a reversible swelling of the hydrogel matrix and a red-shift of the diffraction maxima. Whilst validating the concept of utilising holographic sensors as reporters of glucose concentration, the sensors operated at non-physiological pH values. Subsequent optimisation of the boronic acid moiety enabled the construction of a holographic glucose sensor that responds under physiological pH conditions (Kabilan et al., 2005). Furthermore, in these studies, it was found that the selectivity of the glucose-responsive holographic sensors for glucose in preference to lactate could be tuned by controlling the receptor concentration. Further modification of the polymeric microenvironment with tertiary amine groups has also been shown to improve the selectivity of the sensor for glucose over lactate (Kabilan et al., 2004b).